This goal of this exploratory investigation is to develop a new ELISA test which may rapidly and efficiently detect carcinogens and antioxidants as a means to facilitate cancer prevention, This developmental proposal is based on new novel studies that demonstrated mitochondrial reactive oxygen species (ROS) production as a basic mechanism underlying the pronounced and unique sensitivity of endothelial cells to hyperglycemic stress. The latter induced mitochondrial ROS generation which initiated a pleiotropic cascade resulting in nuclear protein modification as a consequence of oxidative DMA damage. This new signaling pathway involved activation of poly (ADP-ribose) polymerase due to oxidative DNA damage resulting in its unique ADP ribosylation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) inhibiting its function. We shall test the hypothesis that carcinogen induction of ROS in endothelial cells substitutes for hyperglycemic-induction of ROS as the trigger for this signaling pathway. Accordingly, ELISA detection of endothelial GAPDH modification and oxidative DNA adduct formation under normal glucose conditions would provide a new mechanism to measure potential carcinogenicity. Conversely, antioxidants may be detected by reduction in ELISA immunoreactivity as a function of carcinogen exposure or during hyperglycemic stress. Of note, both protein modification and DNA adduct formation may be detected by ELISA in the same cells. Normal human aortic endothelial cells cultured in low and high glucose will be used as the experimental paradigm. ELISA analysis will be performed using anti-(poly-ADP) ribose and anti-8-OH-2'-deoxyguanosine antibodies to quantitate the formation of ADP-ribosylated proteins and the formation of oxidative DNA lesions, respectively. Induction of ROS formation will be tested using arsenic and chromium, human metal carcinogens known to induce oxidative stress. Treatment of cells with ascorbic acid and atorvastatin will be utilized to test the utility of the ELISA as a mechanism to detect antioxidant intervention. En toto, through this exploratory study, we shall define a new mechanism through which the generation of endothelial cell ROS may be used as a detection mechanism for potential carcinogens or therapeutic antioxidants.